BIOPHARMACEUTICS & DRUG DISPOSITION
Biopharm. Drug Dispos. 34: 125–136 (2013)
Published online 24 January 2013 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/bdd.1829

A diclofenac suppository–nabumetone combination therapy forarthritic pain relief and a monitoring method for the diclofenac
binding capacity of HSA site II in rheumatoid arthritis
Nao Setoguchia, Norito Takamurab,*, Ken-ichi Fujitac, Kenji Ogatab, Jin Tokunagab, Toyotaka Nishiod,
Etsuo Chosae, Kazuhiko Arimorie, Keiichi Kawaif, and Ryuichi YamamotogaSecond Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino,
Nobeoka, Miyazaki, 882-8508, Japanb
Second Department of Clinical Pharmacy, Graduate School of Clinical Pharmacy, Kyushu University of Health and Welfare, 1714-1 Yoshino,
Nobeoka, Miyazaki, 882-8508, Japanc
Department of Medical Oncology, International Medical Center, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama, 350–1298, Japand
Shinomiya Pharmacy, NEXUS, Inc., 1-3-57 Shinomiya, Hiratsuka, Kanagawa, 254-0014, Japane
University of Miyazaki Hospital, 5200 Kihara, Kiyotake, Miyazaki, Miyazaki, 889-1692, JapanfSchool of Health Sciences, Faculty of Medical, Kanazawa University, 5-11-80 Kadatsuno, Kanazawa, Ishikawa, 920-0942, JapangFirst Department of Pharmacology, Graduate School of Clinical Pharmacy, Kyushu University of Health and Welfare, 1714-1 Yoshino,
Nobeoka, Miyazaki, 882-8508, Japan

ABSTRACT: Diclofenac suppository, a non-steroidal anti-inﬂammatory drug (NSAID), is usedwidely in rheumatoid arthritis (RA) patients with severe arthritic pain. As the binding percentage of
diclofenac to serum proteins is high, its free (unbound) concentration after rectal administration islow. To increase temporarily the free concentration of diclofenac and to enhance its analgesic effect byinhibiting the protein binding of diclofenac, the analgesic effect of diclofenac was examined before
and after the start of an inhibitor administration to RA patients with insufﬁcient control of arthritic pain,
and the protein binding capacity of diclofenac was evaluated. Binding experiments were performed byultraﬁltration, and arthritic pain was recorded by the face scale. Free fractions of diazepam and diclofenac were augmented by increasing 6-methoxy-2-naphthylacetic acid (6-MNA; the active metabolite of
the NSAID nabumetone) concentrations. The free fraction of diazepam increased after the start of nabumetone administration to RA patients, and arthritic pain relief was observed. These results suggest that
6-MNA has an inhibitory effect on the protein binding of diclofenac and the free fraction of diazepam
can be used to evaluate the binding capacity of diclofenac. It is considered that diclofenac suppository–nabumetone combination therapy and the method for protein binding monitoring by diazepam
can positively beneﬁt RA patients with insufﬁcient control of arthritic pain. Copyright © 2013 John Wiley& Sons, Ltd.
Key words:

rheumatoid arthritis; diclofenac; nabumetone; human serum albumin; protein binding
inhibition

Introduction
The ﬁrst-choice drug for arthritic pain relief inpatients with rheumatoid arthritis (RA) is a non*Correspondence to: Second Department of Clinical Pharmacy,
Graduate School of Clinical Pharmacy, Kyushu University of
Health and Welfare, 1714–1 Yoshino, Nobeoka, 882–8508, Japan.
E-mail: noritotaka@phoenix.ac.jp

Copyright © 2013 John Wiley & Sons, Ltd.

steroidal anti-inﬂammatory drug (NSAID), and adiclofenac suppository is used widely in RA
patients with severe arthritic pain [1,2]. Diclofenac
has the following characteristics: (1) in the case ofrectal administration, it reaches its maximum
plasma concentration quickly (fast-acting) [3]; (2)it is distributed well to synovial membranes andthe synovial ﬂuid [4]; and (3) it has a highReceived 21 September 2012
Revised 1 November 2012
Accepted 30 November 2012

126
inhibitory activity on cyclooxygenase-2 (COX-2)
in human synovial cells [5,6]. Diclofenac suppository is important for reducing severe arthritic pain
in RA patients: however, since the binding percentage of diclofenac to serum proteins is high[7], the free (unbound) concentration of diclofenac
in plasma is low. As a result, the distribution of
diclofenac to tissues is also low. In general, since
the pharmacological effects of a drug depend on
its free concentration [8], only the free drug thatis distributed to tissues reﬂects the effect of the
drug. Therefore, when the relief effect of arthritic
pain is not fully acquired by administering a diclofenac suppository to RA patients, its free concentration needs to be increased.
Our previous study investigated enhancementsin the diuretic effect of furosemide for the purposeof restoring an attenuated response to furosemide
in patients with nephrotic syndrome [9]. The results
showed that bucolome inhibited furosemide bybinding to serum protein, thereby increasing its
free concentration in plasma and urine, and as aresult, restored the diuretic response to furosemide
in rats.
If some drugs can temporarily inhibit thebinding of diclofenac to serum protein using
the previous inhibition method (furosemide–
bucolome–serum protein interaction), the free
concentration of diclofenac in plasma and the
distribution of diclofenac to tissues could be
increased, and as a result, its analgesic effectmay be enhanced. Oral NSAIDs are used
together with a diclofenac suppository in RA
patients with severe arthritic pain [2]. If an oral
NSAID drug can be found that inhibits thebinding of diclofenac to serum protein, more
effective drug treatment than the present treatment for arthritic pain may be possible.
In the present study, in order to identify thebinding protein and binding site of diclofenac,
binding studies of diclofenac to proteins were
performed in human serum. Moreover, anoral NSAID drug was found that inhibits thebinding of diclofenac to serum protein, and theanalgesic effect of diclofenac was veriﬁed
before and after the start of inhibitor administration to RA patients with insufﬁcient control
of arthritic pain. The study examined simultaneously how to evaluate the protein binding
capacity of diclofenac.
Copyright © 2013 John Wiley & Sons, Ltd.

N. SETOGUCHI ET AL.

Materials and Methods
Materials
Each chemical was obtained from the following
sources: pool plasma samples from healthy adults
from Cosmo Bio (Tokyo, Japan); diclofenac from
Novartis Pharma (Tokyo, Japan); diazepam from
Wako Pure Chemicals (Osaka, Japan); warfarinfrom Eisai (Tokyo, Japan); ﬂurbiprofen from
Kaken Pharmaceutical (Tokyo, Japan); ibuprofen
from Tokyo Chemical Industry (Tokyo, Japan);
6-methoxy-2-naphthylacetic acid (6-MNA; theactive metabolite of nabumetone) from SmithKline
Beecham (Tokyo, Japan); and etodolac, humanserum albumin (HSA; essentially fatty acid free),human a1-acid glycoprotein (AGP) and humang-globulins from Sigma-Aldrich (St Louis, MO).
All other chemicals were of analytical grade.

Plasma samples
Blood samples were obtained from three rheumatoid arthritis patients. This study was approvedby the Ethical Committee in the Kyushu University
of Health and Welfare, and informed consent was
obtained before starting the study.
Blood samples were centrifuged immediately
at 1670  g for 10 min, and plasma was stored at
80  C until analysis. The concentrations of albumin were measured by the bromcresol green
method [10], using Cobas Integra 400 plus (Roche
Diagnostics, Basel, Switzerland). The assay kit foralbumin was ALB II (Roche Diagnostics, Basel,
Switzerland).

Identiﬁcation of the binding protein of diclofenac
in human plasma
Identiﬁcation of the binding protein of diclofenac
in human serum was determined with humanplasma and each isolated human serum protein(HSA, AGP, or g-globulins) solution at 25  C
by the following procedures. Human plasma
obtained from each healthy adult was mixed anddiluted with 0.067 M phosphate buffer (pH 7.4)
to adjust the HSA concentration to 600 mM.
Concentrations of isolated HSA (600 mM), AGP(14 mM) and g-globulins (152 mM) were adjusted
by 0.067 M phosphate buffer (pH 7.4). Diclofenac
was added to the diluted plasma and each
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DICLOFENAC–NABUMETONE COMBINATION THERAPY USING MONITORING METHOD

isolated serum protein solution to give a ﬁnal concentration of 3 mM, corresponding to the therapeutic plasma concentrations after a single rectal
administration at a dose of 50 mg [11]. Solutionsof unbound diclofenac were prepared by ultraﬁltration at 2970  g for 15 min at 25  C, using a
Tosoh plastic ultraﬁltration apparatus, Ultracent10 (Tosoh Co., Tokyo, Japan). Adsorption of
diclofenac onto the membrane or apparatus was
negligible.
The free concentrations of diclofenac were determined by the Shimadzu high-performanceliquid chromatography (HPLC) system (Shimadzu,
Kyoto, Japan) consisting of an SPD-20A UV/VIS
detector, LC-10ADvp pump, SIL-10ADvp auto
injector, SCL-10Avp system controller and CTO10Avp column oven, equipped with a LiChrosorb
RP-18 column (7 mm) (Kanto Reagents, Tokyo,
Japan). The HPLC was performed at a ﬂow rate of
1.0 ml/min at 40  C. The eluent was monitored at
275 nm. The mobile phase was a mixture ofacetonitrile, distilled water, methanol and acetic
acid at a ratio of 120: 79: 20: 1 (v/v).
The bound fraction of diclofenac was calculated
as follows:bound fractionðfbÞ ¼½Db ½ Df  þ ½ Db 

(1)where [Df] and [Db] are the unbound and bounddrug concentrations, respectively.

Interaction mode between two ligands at bindingsites of HSA
Binding parameters were determined by ﬁtting
experimental data to the Scatchard equation witha non-linear least squares program (MULTI program) [12]:r¼

m½ Db  X
ni Ki ½Df ¼½Pt 
1 þ K i ½ Df 
i¼1

(2)

where r is the number of moles of bound drug per
HSA molecule, [Pt] is the total HSA concentration,
and Ki and ni are the binding constant and number of binding sites, respectively, for the class ofbinding sites. The simultaneous binding of twoligands was analysed using previously reported
equations [13], as follows:
Copyright © 2013 John Wiley & Sons, Ltd.

rA ¼

127

½Ab 
KA ½Af  þ wKBA KB ½Af ½Bf (3)¼½Pt  1 þ KA ½Af  þ KB ½Bf  þ wKBA KB ½Af ½Bf 

rB ¼

½Bb 
KB ½Bf  þ wKAB KA ½Af ½Bf (4)¼½Pt  1 þ KA ½Af  þ KB ½Bf  þ wKBA KB ½Af ½Bf 

where KA and KB are the binding constants ofligands A and B, respectively, [Af] and [Bf] are
the respective free concentrations of ligands A
and B, and [Ab] and [Bb] are the bound concentrations of ligands A and B, respectively. w is a
coupling constant, KBA is the binding constant ofligand A in the presence of ligand B, and KAB isthe binding constant of ligand B in the presenceof ligand A. Theoretical values of w were calculated with these equations. The interaction modeof ligands on a macromolecule can be evaluatedfrom the sign and magnitude of the value of w.
For example, if ligands A and B are independently bound to HSA, w is equal to 1. The results
w > 1 and 0 < w < 1 indicate cooperative andanti-cooperative interactions between ligands.
Competitive displacement between ligands isindicated by w = 0. In this analysis, r < 0.31 wasused to suppress the contribution of a low-afﬁnity
binding site.
Solutions of unbound diclofenac, diazepam,
warfarin and 6-MNAwere prepared by ultraﬁltration
at 2970  g for 10 min at 25  C with Ultracent-10.
As well as diclofenac, the adsorption of diazepam,
warfarin and 6-MNA onto the membrane or apparatus was negligible.
Free concentrations of diclofenac, diazepam,
warfarin and 6-MNA were determined by the
HPLC system described above. The mobile phase
was a mixture of acetonitrile, distilled water,
methanol and acetic acid. The ratios were
100:99:20:1 (v/v) for diclofenac, 110:89:20:1 (v/v)
for diazepam and 80:119:20:1 (v/v) for warfarin
and 6-MNA.

Inﬂuence on the HSA binding of diclofenac by
NSAIDs
Non-steroidal anti-inﬂammatory drugs including
ﬂurbiprofen, etodolac, ibuprofen and 6-MNA(active metabolite of nabumetone) were used as
inhibitors of diclofenac binding to HSA, since these
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128

N. SETOGUCHI ET AL.

NSAIDs have been reported to bind to site II of
HSA [14,15] and their therapeutic plasma concentrations are high [16–19]. The following procedures
were performed at 25  C. Plasma obtained fromeach healthy adult was mixed and diluted with
0.067 M phosphate buffer (pH 7.4) to adjust the
HSA concentration to 600 mM. Diclofenac wasadded to diluted plasma to give a ﬁnal concentration of 3 mM. Flurbiprofen, etodolac, ibuprofen or
6-MNA was further added to give a ﬁnal concentration of 30, 50, 100 or 200 mM after the addition of
diclofenac. These concentrations corresponded totherapeutic plasma levels after single or continuous
oral administrations (40 mg for ﬂurbiprofen,
200 mg for etodolac, 200 mg for ibuprofen and
800 mg for nabumetone) [16–19]. Solutions of unbound diclofenac were prepared by ultraﬁltration
at 2970  g for 15 min at 25  C with Ultracent-10.
Free concentrations of diclofenac were determined by the HPLC system described above.
The mobile phase was a mixture of acetonitrile,
distilled water, methanol and acetic acid at a ratioof 110:89:20:1 (v/v).The free fraction of diclofenac
was calculated as follows:
free fraction ðfuÞ ¼½ Df 
½Df  þ ½Db (5)

Establishment of a monitoring method for thein vivo HSA binding of diclofenac
An ex vivo monitoring method to evaluate thein vivo inhibitory effect of 6-MNA on the HSA binding of diclofenac was established by in vitro experiments as follows. The occupancy of the HSA site IIwith or without the addition of 6-MNA wasmonitored with diazepam. All procedures were
performed at 25  C. Plasma obtained from eachhealthy adult was mixed and diluted with 0.067 M
phosphate buffer (pH 7.4) to adjust the HSA
concentration to 400 mM. Plasma containing 500 or
600 mM HSA was prepared by adding HSA powder
to diluted plasma. Diclofenac and diazepam wereadded to each plasma to give a ﬁnal concentrationof 20 mM. Moreover, the ﬁnal concentration of
6-MNA added to each plasma ranged from 0 to
600 mM (6-MNA/HSA= 0, 0.25, 0.5, 0.75, 1.0). Solutions of unbound diclofenac and diazepam wereprepared by ultraﬁltration at 2970  g for 15 min
at 25  C with Ultracent-10.
Copyright © 2013 John Wiley & Sons, Ltd.

The free concentrations of diclofenac and diazepam were determined by the HPLC systemdescribed above. The mobile phase consisted ofacetonitrile, distilled water, methanol and acetic
acid at a ratio of 110:89:20:1 (v/v) for diclofenac
and 100:99:20:1 (v/v) for diazepam.
Free fractions of diclofenac and diazepam were
calculated using Equation (5).

Evaluation of the HSA binding capacity of diclofenac and arthritic pain by 6-MNA in RA patients
The binding capacity of diclofenac to site II of
HSA in the absence and presence of 6-MNA wasmonitored by diazepam as established above.
Diazepam was added to the plasma of each patient
before and after the start of nabumetone administration to give a concentration of 20 mM in a ﬁnal
volume of 0.5 ml, and two free fractions were compared. Blood collection after the start of nabumetone administration was performed from day 4
after the start of nabumetone administration where
the plasma concentration of 6-MNA had alreadyreached a steady-state [19].
Solutions of unbound diazepam were preparedby ultraﬁltration at 2970  g for 15 min at 25  Cwith Ultracent-10.
Free concentrations of diazepam were determined by the HPLC system described above.
The mobile phase was a mixture of acetonitrile,
distilled water, methanol and acetic acid at a ratioof 100:99:20:1 (v/v).
Free fractions of diazepam were calculated using
Equation (5).
Arthritic pain caused by RA was recorded by theface scale. The face scale shows various faces from alaughing face, corresponding to a state of no pain,
to a very unhappy face, corresponding to a stateof worst pain. Faces are arranged in enhancing
order of pain and are numbered from 1 to 10, with
1 representing ‘no pain’ and 10 representing ‘worst
pain’. Arthritic pain was evaluated just before diclofenac suppository administration for 5 days before
and after the start of nabumetone administration.
Arthritic pain in patient 2 was evaluated just before
diclofenac suppository administration at 17:00.
Assessment of arthritic pain for 5 days after the startof nabumetone administration was performed fromday 4 after the start of nabumetone administration,
as well as at the time of blood collection timing.
Biopharm. Drug Dispos. 34: 125–136 (2013)
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DICLOFENAC–NABUMETONE COMBINATION THERAPY USING MONITORING METHOD

Statistical analysis
Statistical differences were assessed using Dunnett’s
test for multiple comparisons after a one-way analysis of variance (ANOVA), with a probability levelof p < 0.01 regarded as signiﬁcant.

Results
Identiﬁcation of the binding protein of diclofenac
in human plasma
To evaluate the binding capacity of diclofenac to
HSA, AGP and g-globulins, the free concentrationof diclofenac was measured and its binding fraction
calculated. Diclofenac strongly bound to HSA andthe binding fraction was 99.99%. On the other hand,binding fractions to AGP and g-globulins were low
at 17.75% and 18.71%, respectively (Figure 1). Thisin vitro study indicated that diclofenac strongly
binds to HSA in human plasma.

Identiﬁcation of the HSA binding site of
diclofenac
To identify the HSA binding site of diclofenac, atypical HSA site I binding drug, warfarin, and a
HSA site II binding drug, diazepam, were used.
The interaction mode between diclofenac and thesite-speciﬁc probe was analysed according to themethod of Kragh-Hansen [20]. Figure 2 shows

129

the results of a quantitative analysis of mutual
displacement between diclofenac and warfarin or
diazepam. The kinetic binding constant (n1K1) ofdiclofenac, diazepam and warfarin on binding to
HSA were determined to be 3.8  106 M-1,
1.4  106 M-1 and 2.1  105 M-1, respectively (datanot shown). Taking their parameters into consideration, a 30 mM concentration of these ligands will
inhibit the binding to HSA. In this condition, thebinding isotherm of diclofenac in the presence ofdiazepam, and vice versa, was fairly close to thetheoretical curve that assumed competition between these two ligands at a common site (Figure 2,
A and B). On the other hand, the binding isothermof diclofenac in the presence of warfarin, and viceversa, was fairly close to the theoretical curve thatassumed independence between these two ligands
at a common site (Figure 2, C and D).

Inﬂuence of NSAIDs on the HSA binding of
diclofenac
To identify which oral NSAIDs inhibit the bindingof diclofenac to site II of HSA, the inhibitory effectof ﬂurbiprofen, etodolac, ibuprofen and 6-MNA
were examined at therapeutic plasma concentrations. The results indicated that 6-MNA signiﬁcantly inhibited the HSA binding of diclofenac,
and there were no signiﬁcant differences in ﬂurbiprofen, etodolac and ibuprofen (Figure 3).

Interaction mode between diclofenac and 6-MNA
at binding sites of HSA

Figure 1. Binding of diclofenac to human serum, HSA, AGP andg-globulins by ultraﬁltration at pH 7.4 and 25  C. The following
concentrations were used: [serum] (as HSA), 600 mM; [HSA],
600 mM; [AGP], 14 mM; [g-globulins], 152 mM; and [diclofenac],
3 mM. Each column is the mean of three experiments  SD
Copyright © 2013 John Wiley & Sons, Ltd.

The type of binding inhibition was analysed
according to the method of Kragh-Hansen [20] because 6-MNA at therapeutic plasma concentrations
inhibited diclofenac binding to site II of HSA.
Figure 4 shows the results of a quantitative analysis
of mutual displacement between diclofenac and
6-MNA. The kinetic binding constant (n1K1) ofdiclofenac and 6-MNA on binding to HSA were
determined to be 4.2  106 M-1 and 1.2  107 M-1,
respectively (data not shown). Taking these parameters into consideration, a 30 mM concentrationof these ligands will inhibit the binding to HSA. In
this condition, the binding isotherm of diclofenac
in the presence of 6-MNA, and vice versa, wasfairly close to the theoretical curve that assumed
competition between these two ligands at acommon site.
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130

N. SETOGUCHI ET AL.
0.4

0.4

(A)

0.3

0.3

0.2

0.2

0.1

0.1

0.0

(B)

0.0
0

0.4

0.1
0.2
[Diclofenac] f (µM)

0.3

0

0.4

(C)

0.3

0.3

0.2

0.2

0.1

0.1

0.0
0

0.05
0.1
[Diclofenac] f (µM)

0.15

0.5
[Diclofenac] f (µM)

1

(D)

0.0
0

0.5
1
[Warfarin] f (µM)

1.5

Figure 2. Binding of diclofenac in the presence of diazepam (A) and vice versa (B), warfarin (C) and vice versa (D) to HSA at pH 7.4
and 25  C. (A) Binding of diclofenac (15–27.5 mM) to HSA (90 mM) in the presence of diazepam (30 mM); (B) Binding of diazepam(15–27.5 mM) to HSA (90 mM) in the presence of diclofenac (30 mM); (C) Binding of diclofenac (15–27.5 mM) to HSA (90 mM) in thepresence of warfarin (30 mM); (D) Binding of warfarin (15–27.5 mM) to HSA (90 mM) in the presence of diclofenac (30 mM). ● Experimental values; ------- Theoretical curve assuming competitive binding; –––––––, Theoretical curve assuming independent binding.
All theoretical curves were constructed using n1 and K1 values [diclofenac, n1K1 = 3.8  106 M-1; diazepam, n1K1 = 1.4  106 M-1;
warfarin, n1K1 = 2.1  105 M-1]

Establishment of a monitoring method for thein vivo HSA binding of diclofenac

Free fraction of diclofenac

0.007
0.006
0.005
0.004
0.003
0.002
0.001
0
controlwithflurbiprofen
30 µM

with
etodolac
50 µM

with
ibuprofen
100 µMwith
6-MNA
200 µM

Figure 3. Inﬂuence on diclofenac HSA site II binding at the clinically highest blood levels by various NSAIDs binding to site
II of HSA. The following concentrations were used: [serum](as HSA), 600 mM; [diclofenac], 3 mM; [ﬂurbiprofen], 30 mM;[etodolac], 50 mM; [ibuprofen], 100 mM; and [6-MNA], 200 mM.
Each column is the mean of three experiments  SD. **p < 0.01
signiﬁcantly different from the control
Copyright © 2013 John Wiley & Sons, Ltd.

As a monitoring method to evaluate the binding
capacity of diclofenac to site II of HSA in theabsence and presence of 6-MNA in RA patients,
free fractions were compared using free and total
concentrations of diclofenac before and after thestart of nabumetone administration. However,these total concentrations before and after the startof nabumetone administration have to be equal[21]. It is very difﬁcult to obtain blood that contains the same total concentrations of diclofenac
because time-dependent changes in plasma concentrations vary widely according to changes in
condition (including physical condition andendogenous substances) on the day. An attempt
was made to establish a monitoring method that
evaluates the binding capacity of diclofenac to site
II of HSA using diazepam, which binds to thesame HSA site II as diclofenac. It is considered
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DICLOFENAC–NABUMETONE COMBINATION THERAPY USING MONITORING METHOD
0.4

0.4

(A)

0.3

0.3

0.2

0.2

0.1
1

0.1

0.0

0

0.1

0.2

0.0

0.3

(B)

0

0.05

[Diclofenac]] f (µM)

0.1

0.15

[6-MNA] f (µM)

Figure 4. Binding of diclofenac in the presence of 6-MNA (A) and vice versa (B) to HSA at pH 7.4 and 25  C. (A) Binding of diclofenac (15–27.5 mM) to HSA (90 mM) in the presence of 6-MNA (30 mM); (B) Binding of 6-MNA (15–27.5 mM) to HSA (90 mM) in thepresence of diclofenac (30 mM). ● Experimental values; --------- Theoretical curve assuming competitive binding; –––––––, Theoretical curve assuming independent binding. All theoretical curves were constructed using n1 and K1 values [diclofenac, n1K1 = 4.2
 106 M-1; 6-MNA, n1K1 = 1.2  107 M-1]

that it will be possible correctly to evaluate thebinding capacity of diclofenac to site II of HSA
by comparing these free fractions after adding
diazepam at a ﬁxed concentration to the plasmaof each patient obtained by collecting blood before
and after the start of nabumetone administration.
Moreover, it will be possible to simply and speedily perform this evaluation because, in this monitoring method, it is not necessary to measure
total diazepam concentrations in plasma byadding the same concentration of diazepam to theplasma of patients before and after the start of nabumetone administration. Diclofenac contained in theplasma of patients does not affect the binding ofdiazepam to site II of HSA because the therapeuticplasma concentration of diclofenac (approximately
3 mM) is markedly lower (1/200–1/133) than that

0.006

Evaluation of the HSA binding capacity of
diclofenac and arthritic pain by 6-MNA in RA
patients
Table 1 shows the information of three RA
patients (two inpatients and an outpatient) who
tried to increase free concentrations of diclofenac.
Diclofenac suppository was administered to each

0.08

(A)

Free fraction of diazepam
F

Free fracction
n off diclofenac

0.007

of HSA (400–600 mM) (data not shown). To conﬁrm
whether the binding of diazepam as well as diclofenac to site II of HSA was inhibited by 6-MNA, thefree fraction of diazepam was compared with thatof diclofenac at [6-MNA/HSA] = 0, 0.25, 0.5, 0.75
and 1.0. As a result, free fractions of diazepam anddiclofenac were augmented by increasing 6-MNA
concentrations (Figure 5).

0.005
0.004
0.003
0.002
0.001
0.000

0.07

(B)

0.06
0.05
0.04
0.03
0.02
0.01
0.00

0

0.25

0.5

0.75

6-MNA/HSA

1.0

0

0.25

0.5

0.75

1.0

6-MNA/HSA

Figure 5. Effect of 6-MNA on the free fraction of diclofenac and diazepam to site II of HSA. The following concentrations were used:[serum] (as HSA), ▲ 400 mM; ♦ 500 mM; ■ 600 mM; [diclofenac], 20 mM; and [diazepam], 20 mM
Copyright © 2013 John Wiley & Sons, Ltd.

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Copyright © 2013 John Wiley & Sons, Ltd.

Male, 59

Female, 73

Female, 47

Patient 1(inpatient)

Patient 2(inpatient)

Patient 3(outpatient)

Gender, age

Rheumatoid
arthritis

Arthralgia

Occipitoaxial luxation(occipitoaxial pexis), numbness
in the hands, pain in the inferior limb

Atlantoaxial subluxation(excision of vertebrae cervicales
arcus vertebrae, occipital pexis),
decrease in myodynamia,
numbness in the upper limbs,
lumbagoTable 1. Rheumatoid arthritis patient information

Diclofenac suppository
25 mg daily (06:00)
Etodolac tablet 200 mg
twice daily (07:30 and 19:30)

Diclofenac suppository
50 mg daily (05:00)
Diclofenac suppository
25 mg daily (17:00)

Diclofenac suppository
25 mg daily (17:00)

Without
nabumetone

Withnabumetone

Diclofenac suppository
25 mg daily (06:00)
Nabumetone tablet 800 mg daily (19:30)

Diclofenac suppository
25 mg daily (05:00)
Diclofenac suppository
12.5 mg daily (17:00)
Nabumetone tablet 800 mg daily (13:00)

Diclofenac suppository 12.5 mg
daily (17:00) Nabumetone
tablet 800 mg daily (13:00)

Medication for painor fever control

132
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133

DICLOFENAC–NABUMETONE COMBINATION THERAPY USING MONITORING METHOD

RA patient in consideration of the degree ofarthritic pain and the time at which pain was
experienced. It has been reported that the time
to maximum plasma concentration (Tmax) of
6-MNA after oral administration of nabumetone
is 4.0 h [19] in humans. To obtain the maximum
inhibitory effect of 6-MNA on the binding of
diclofenac to site II of HSA, nabumetone(800 mg) was given 4.0 h before diclofenac suppository administration. However, the administration time of nabumetone according topatient’s life style was considered, as it was
difﬁcult to administer nabumetone 4.0 h before
diclofenac suppository administration. Nabumetone was administered only once a day because
6-MNA has a long half-life (t1/2) of approximately
20.5 h [19]. In patient 1, since the diclofenac suppository needed to be administered at 17:00,
nabumetone was given after lunch (13:00), 4.0 h
before diclofenac suppository administration.
Patient 2 needed an administration of diclofenac
suppository twice a day, and her arthritic pain
was especially severe in the evening. Therefore,
nabumetone was administered after lunch(13:00), 4.0 h before diclofenac suppository administration (17:00), to obtain the maximum
inhibitory effect in the evening. In patient 3, since
the diclofenac suppository needed to be administered at 06:00, nabumetone should have been
given at 02:00, 4.0 h before the diclofenac suppository administration. However, nabumetone wasadministered after dinner (19:30), the day before
the diclofenac suppository administration because
it was difﬁcult to administer nabumetone at 02:00.
The increase in the free fraction of diazepam after
the start of nabumetone administration in patients
1, 2, and 3 was 3.65 times (0.062/0.017), 2.15 times(0.043/0.020) and 1.78 times (0.032/0.018), respectively (Table 2). Arthritic pain relief was simultaneously observed in all patients. The HSA
concentrations remained unchanged in patients 1
and 2, and increased in patient 3 who had nutritional recovery by increasing appetite duringarthritic pain relief.

Discussion
In the present study, it was shown that diclofenac
strongly bound to site II of HSA (Figures 1 and 2).
On the basis of these results, drugs that inhibit thebinding of diclofenac to site II of HSA were investigated, and, as a result, 6-MNA, the active metabolite of the NSAID nabumetone, inhibited the HSA
binding of diclofenac at a therapeutic plasma concentration (Figures 3 and 4). The degree of inhibition of diclofenac was enhanced with increasing
concentrations of 6-MNA, HSA (site II) binding ofdiazepam to the same HSA site II as diclofenac
was also inhibited by 6-MNA, and the degree of inhibition was enhanced with increasing concentrations of 6-MNA (Figure 5). These results suggestthat diazepam can evaluate the binding capacityof diclofenac to site II of HSA (Figure 6). On the basis of the preliminary examination results, the binding capacity of diclofenac to site II of HSA and thedegree of arthritic pain in diclofenac suppository–
nabumetone combination therapy were evaluatedin three RA patients. As a result, marked increases
in the free fraction of diazepam and arthritic pain
relief were observed after the start of nabumetone
administration in all patients (Table 2).
The diclofenac suppository demonstrated ananalgesic effect with a single dose and wasTable 2. Diagnosis of HSA site II binding property in rheumatoid arthritis patients
Blood collection time

Free fraction of diazepam

HSA (mM)

Face scale

Patient 1 (inpatient)

Without nabumetone
With nabumetone

17:30
17:50

0.017
0.062

433
468

7.6  0.5
4.7  0.2a

Patient 2 (inpatient)

Without nabumetone
With nabumetone

05:45
17:30

0.020
0.043

519
513

7.0  0.9
4.6  0.7b

Patient 3 (outpatient)

Without nabumetone
With nabumetone

11:00
10:36

0.018
0.032

414
511

8.0  0.0
4.0  0.0c

p < 0.01 vs before inhibition for patient 1.
p < 0.01 vs before inhibition for patient 2.
cp < 0.01 vs before inhibition for patient 3.
abCopyright © 2013 John Wiley & Sons, Ltd.

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134

N. SETOGUCHI ET AL.
Diazepam addition(Diazepam final
concentration 20 µM))

Ultrafiltration(2970 g, 15 min)
0.5 mlFiltrate S1 (T1)

PlasmaS1 (T1)Pl(without nabumetone)

S2
Free fractionof diclofenac

F e fraction
Freof diazepam

S2

S1
T1

T2

S1
T1

Time

T2
Time

0.5 mlFiltrate S2 (T2)
Plasma S2 (T2)(witi h nabumetone)

Frree fractionof diazepam

Ultrafiltration(2970 g, 15 min)

S2
S1
T1

T2
Time

Frree fraction
off diclofenac

orDiazepam addition(Diazepam final
concentration 20 µM)

S2
S1
T1

T2
Time

Figure 6. Monitoring method for the binding capacity of diclofenac to site II of HSA by diazepam in RA patients. Diazepam wasadded to the patient’s plasma sample 1 (S1) and sample 2 (S2) of 0.5 ml before (T1) and after (T2) the start of nabumetone administration to give a ﬁnal concentration of 20 mM. Each ﬁltrate was obtained by ultraﬁltration. Free fractions of diazepam of S1 and S2
were compared, and the degree of increase in the free fraction of diazepam from T1 to T2 can be calculated. The degree of increase in
the free fraction of diclofenac from T1 to T2, i.e. the degree of binding capacity of diclofenac to HSA site II before (T1) and after (T2)the start of nabumetone administration is predicted by the free fraction of diazepam

distributed well to synovial membranes and thesynovial ﬂuid [4]. Therefore, there is a high probability that arthritic pain relief is observed evenwith transient increases in the free concentrationof diclofenac in plasma. For this reason, because
diclofenac has very high inhibitory activity on
COX-2 compared with nabumetone [22], the analgesic effect of the diclofenac suppository wasmuch higher than that of nabumetone. Therefore,
it is considered that nabumetone gives little arthritic pain relief to RA patients with insufﬁcient
control of arthritic pain from a diclofenac suppository. In fact, arthritic pain was signiﬁcantly reduced
when changing from etodolac to nabumetone in patient 3, although the inhibitory effect of etodolac(400 mg/day) on COX-2 was greater than that ofnabumetone (800 mg/day) (Tables 1 and 2). Therefore, it is considered that the pain relief was due totransient increases in the free concentration of diclofenac by 6-MNA in plasma.
The therapeutic plasma concentrations of
6-MNA (250 and 266 mM at 4 h after administrationfor 3 and 42 days, respectively) [19] corresponded
to about one-half of the HSA concentrations
Copyright © 2013 John Wiley & Sons, Ltd.

468–513 mM after the start of nabumetone administration (Table 2). As shown in Figure 3, since 6MNA (200 mM), corresponding to one-third theconcentration of HSA (600 mM) in plasma, signiﬁcantly inhibited the binding of diclofenac to site IIof HSA, it is considered that the therapeutic plasma
concentration of 6-MNA is a level sufﬁcient as abinding inhibitor of site II of HSA. Furthermore,nabumetone is available early after administration
because the plasma concentration of 6-MNA after
nabumetone administration reaches a sufﬁciently
high concentration as the binding inhibitor ofsite II of HSA. In addition, since 6-MNA has a longt1/2 [19], it is considered that 6-MNA sufﬁciently
inhibits the binding of diclofenac to site II of
HSA even if the timing of diclofenac suppository
administration was shifted too far from the Tmaxof 6-MNA.
In general, if the free concentration of a drug ismarkedly increased by inhibiting protein binding,its elimination from blood is increased and its
plasma concentration is decreased [23,24]. As aresult, since accumulation of the drug does nottake place, it is expected that the occurrence ofBiopharm. Drug Dispos. 34: 125–136 (2013)
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DICLOFENAC–NABUMETONE COMBINATION THERAPY USING MONITORING METHOD

side effects should be low. When the metabolism
of a drug is also inhibited, its free concentration
can be expected to increase due to higher plasma
concentrations. However, since the inhibition of
drug metabolism suppresses elimination of thedrug, plasma concentrations increase markedly.
As a result, the occurrence of side effects may behigh. The inhibition of diclofenac metabolism does
not occur in diclofenac suppository–nabumetone
combination therapy because nabumetone and6-MNA do not inhibit CYP2C9 that is the ratelimiting enzyme in the metabolic clearance of diclofenac [19,25]. Therefore, since it has been suggested
that increases in free plasma diclofenac concentrations occurred due to protein binding inhibition, it
is considered that the occurrence of side effects
should be low. In fact, no side effects were observedin the three patients.
The binding capacity of diclofenac to site II of
HSA in diclofenac suppository–nabumetone combination therapy was evaluated. In this evaluation
diazepam was added to the plasma of eachpatient before and after the start of nabumetone
administration at the same ﬁnal concentration,and these free fractions of diazepam were compared (Figure 6). Blood collection after the start ofnabumetone administration is recommended at atime between immediately after administration of
the diclofenac suppository and the Tmax. It is considered that plasma concentrations of 6-MNA witha long t1/2, that is, the inhibitory effect on thebinding of diclofenac to HSA, remains mostly
unchanged in that time range because diclofenac
reaches Cmax immediately after rectal administration. After the start of nabumetone administration,
blood was collected from patients 1 and 2 andwas evaluated at the optimal time for the maximum
inhibitory effect on the binding of diclofenac to
HSA. The increase in the free fraction of diazepam
after the start of nabumetone administration was
3.65 and 2.15 times, respectively, proving that the
inhibitory effect of 6-MNA on the binding of diclofenac to HSA was strong. Since there were large
differences in the inhibitory effect between patients
1 and 2, it is possible to conjecture that there werelarge individual differences in the Cmax of nabumetone between the two patients. On the other hand,in the case of the outpatient, it was difﬁcult tocollect blood at the optimal time. Patient 3 was an
outpatient, and blood collection after the start of
Copyright © 2013 John Wiley & Sons, Ltd.

135

nabumetone administration was performed at
10:36 (consultation time), 4 h 36 min after the diclofenac suppository administration (06:00). It seems
that the plasma concentration of 6-MNA was lower
than that immediately after administration of the
diclofenac suppository because the blood collection
point may have passed approximately 15 h after
nabumetone administration (19:30). Therefore, it
seems that the maximum inhibitory effect on
diclofenac binding to HSA was higher than that
(1.78 times) in the blood that was collected andevaluated. Thus, even if blood collection cannot be
performed at the optimal time, the inhibitory effectof 6-MNA on HSA binding immediately afteradministration of diclofenac suppository can bepredicted using the administration time, Tmax andt1/2 of nabumetone. Moreover, the monitoring
method can obtain useful information by comparing the free fraction of diazepam between patients
before the start of nabumetone administration.
For example, although the concentration of HSA(519 mM) in patient 2 before the start of nabumetone
administration was higher than that (433 mM) in
patient 1, the free fraction of diazepam (0.020) inpatient 2 was higher than that (0.017) in patient 1.
It can be predicted that there was a difference inthe microenvironment around the HSA site II[26,27] or in the molecular structures [28,29]
between the two patients.
In conclusion, it was shown that when the
diclofenac suppository was administered at atherapeutic plasma concentration of 6-MNA, thebinding to site II of HSA was inhibited and theanalgesic effect of diclofenac increased because ofthis inhibitory effect. The diclofenac suppository–
nabumetone combination therapy can be performed safely only by considering the combinationof drugs for RA and the administration timing.
Furthermore, it was found that the free fraction ofdiazepam can be used to monitor the inhibitory
effect of 6-MNA on the HSA binding of diclofenac.
The monitoring method can also predict the microenvironment around the HSA site II [26,27] or themolecular structure [28,29] by comparing the binding capacity to site II of HSA between patients. It isconsidered that this monitoring method is the optimal approach for evaluating binding capacities inclinical practice because there is very little patientburden and it is possible to perform the evaluation
simply and speedily. It is considered that diclofenac
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136
suppository–nabumetone combination therapy andthe method for monitoring HSA binding by diazepam described in the present study can positively
beneﬁt rheumatoid arthritis patients withinsufﬁcient control of arthritic pain.

Conﬂicts of Interest
The authors have no conﬂicts of interest.

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